Ewing sarcoma family tumors (ET) are highly aggressive bone and soft tissue tumors for which novel therapies are desperately needed. Almost all ET express the fusion oncogene EWS-FLI1, a transcription factor whose precise mechanism of action is unknown. Unfortunately, the cellular origin of ET remains elusive and this has greatly impeded research efforts aimed at identifying novel therapeutic targets. Recent data from our lab and others suggest the ET arise from malignant transformation of neural crest and/or mesenchymal stem cells. The polycomb gene BMI-1 is highly expressed by many human cancers and functions to promote stem cell self-renewal, in large part through CDKN2A repression. We have recently shown that BMI-1 acts as an oncogene in ET but that this is mediated in a CDKN2A-independent manner. Importantly, our data suggest that BMI-1-mediated changes in cell adhesion may promote tumor formation. In this proposal we will test the hypothesis that ET arise from neural crest stem cells as a result of oncogenic cooperation between EWS-FLI1 and BMI-1. It is our goal to define the mechanism of BMI-1 oncogenic activity in ET maintenance (Aim 1) and initiation (Aim 2 & 3). Using in vitro and in vivo assays we will determine if ET cell self-renewal is dependent on BMI-1. We will also investigate the impact of BMI-1 over-expression on tumor engraftment in local and metastatic sites. By assessing the consequences of EWS-FLI1 expression in cells that express variable levels of BMI-1, we will define the molecular mechanisms of EWS-FLI1/BMI-1 cooperation and determine if these oncogenes are together necessary and sufficient to induce malignant transformation of primary stem cells. Our preliminary studies have revealed that polycomb gene targets are frequently hyper-methylated in ET cells compared to untransformed neural crest and mesenchymal stem cells. We will use a highly innovative model of human embryonic stem cell-derived neural crest stem cell differentiation combined with an inducible EWS- FLI1 expression vector to determine if activation of the fusion oncogene leads to aberrant polycomb-mediated silencing of developmental pathways. The importance of BMI-1 and polycomb activity in cancer biology is now well established. Using ET as a model this proposal will generate novel insights into BMI-1 function in human tumor initiation and progression. Identification of BMI-1 -modulated pathways will generate novel targets for therapeutic intervention that can be exploited in the many human cancers that have effectively hijacked BMI-1 activity.